The long-term goal of the proposed research project is to understand the molecular mechanisms of cellular responses to the unique tumor microenvironment (low glucose concentration, chronic hypoxia, and low extracellular pH). We previously observed that glucose deprivation increases the intracellular concentration of hydroperoxide. It also activates the SAPK (stress activated protein kinase) signal transduction pathway. In this grant proposal, we postulate that glutaredoxin (GRX), a redox-regulatory protein, recognizes the metabolic oxidative stress and triggers the ASK1-MEK-MAPK signal transduction pathway. The guiding hypothesis is that glucose deprivation raises the intracellular level of reactive oxygen species (ROSs) and increases the level of oxidized glutathione. GRX, which contains two redox-active half-cystine residues (Cys-Pro-Tyr-Cys) in an active center, recognizes metabolic oxidative stress through catalysis of thiol-disulfide interchange reactions with oxidized molecules such as oxidized glutathione. The oxidized GRX dissociates from ASK1 (apoptosis signal regulating kinase 1). The dissociation of GRX from ASK1 results in the activation of ASK1 and subsequently activates the ASK1-MEK-MAPK signal transduction pathway. The specific aims of this project are to examine (1) how GRX recognizes the metabolic oxidative stress, (2) the role of GRX in the ASK1-MEK-MAPK signal transduction pathway, (3) cooperation between GRX and thioredoxin (TRX) to regulate ASKI activation during glucose deprivation. The proposed studies for the first aim employ site-directed mutagenesis to create a point mutant at the redox-active site as well as at other cysteine residues. These studies will illustrate how half-cystine residues are involved in the recognition of metabolic oxidative stress. The second aim will employ molecular genetics and biochemical techniques to elucidate the involvement of GRX in the ASK1-MEK-MAPK signal transduction pathway. The third aim will use the immune complex kinase assay to assess the effect of intracellular glutathione deprivation, extracellular oxidized glutathione treatment, inhibition of GRX or TRX gene expression on ASK1 activation. We believe that investigating the mechanisms of metabolic oxidative stress responses in tumor cells will provide insight into how tumor cells recognize metabolic oxidative stress and initiate signal transduction.